The catecholamine, norepinephrine (NE) governs a spectrum of physiologic processes from vasoconstriction and heart rate to attention and motivation. NE signaling is dynamically regulated by a diverse set of macromolecules including NE transporters (NETs). NETs expressed in the CNS and periphery are important targets for tricyclic antidepressants and psychostimulants. Altered NE transport is documented in cardiovascular diseases and brain disorders. Second messenger linked kinase mediated regulation of human genetic variants of NET is altered in some cardiovascular phenotypes signifying the search for underlying mechanisms of NE transport regulation. NET contains putative phosphorylation sites for several kinases including protein kinase C (PKC). Recently, we demonstrated that, in rat placental trophoblasts, PKC activation enhances native NET internalization via lipid rafts and stimulates transporter phosphorylation. Following the studies on native NET regulation, we directed our studies to explore the role of NET phosphorylation in NE transport regulation. Using a human placental trophoblast cell line expressing hNET (HTR-hNET cells), we demonstrate that a double mutation at a predicted PKC phosphorylation motif of hNET prevents neurokinin 1 receptor (NK1R)/PKC-mediated transporter regulation and phosphorylation. Excessive neurokinin secretion is linked to pre-eclampsia, and thus, the regulation of placental NET by NK1R activation suggests physiological relevance of such regulation in the maintenance of a normal pregnancy. Based on these observations, we propose to test a specific hypothesis that NET phosphorylation is required for NE transport regulation in two separate Specific Aims. In Specific Aim I, we will identify the signals, specific sites and motifs involved in NK1R/PKC regulated NET phosphorylation and expression to explore the relationship between NET phosphorylation and NE transport. Specific Aim II will test the hypothesis that NK1R/PKC regulated NET phosphorylation and interaction with transporter-associated proteins occur in lipid rafts, and that this association establishes transporter distribution and function in an activity-dependent manner to cope with the demands in the milieu of incoming signals. The results from this research will provide information that could be of use in the development of new therapeutic strategies aimed at NE transport regulation in the treatment of both cardiovascular diseases and brain disorders.